SUMMARY

In cicadas, the tympanum is anatomically intricate and employs complex
vibrations as a mechanism for auditory frequency analysis. Using microscanning
laser Doppler vibrometry, the tympanal mechanics of Cicada orni can
be characterized in controlled acoustical conditions. The tympanum of C.
orni moves following a simple drum-like motion, rather than the
travelling wave found in a previous study of Cicadatra atra. There is
a clear sexual dimorphism in the tympanal mechanics. The large male tympanum
is unexpectedly insensitive to the dominant frequency of its own calling song,
possibly a reflection of its dual purpose as a sound emitter and receiver. The
small female tympanum appears to be mechanically sensitive to the dominant
frequency of the male calling song and to high-frequency sound, a capacity
never suspected before in these insects. This sexual dimorphism probably
results from a set of selective pressures acting in divergent directions,
which are linked to the different role of the sexes in sound reception and
production. These discoveries serve to indicate that there is far more to be
learnt about the development of the cicada ear, its biomechanics and
evolution, and the cicada's acoustic behaviour.

INTRODUCTION

The production and reception of sound plays an important role in the life
history of many insect species. For example, pair formation is ruled by the
production and reception of sound between partners, and the orientation
towards a prey or away from a predator can be elicited by acoustic cues
(Greenfield, 2002). The
success of such behaviour depends on the activation of complex auditory
organs, either antennae working as near-field particle velocity detectors, or
tympanal ears acting as far field pressure receivers
(Robert and Göpfert,
2002; Robert and Hoy,
2007). In the latter case, the receiving organ is typically
organized around a thin tympanal membrane (TM) made of cuticle, backed with
air-filled tracheal sacs, and a set of sensory neurons connected to glial and
support cells (for reviews, see Yager,
1999b; Yack,
2004). Such hearing organs have independently evolved in seven
insect orders showing different degrees of development and organization
(Hoy and Robert, 1996).
Whatever the complexity of the hearing system is – from the `cyclopean'
ear of the praying mantis (Yager and Hoy,
1986), to the highly innervated pair of conspicuous cicada ears
(Fonseca et al., 2000) –
differences between male and female have been very rarely reported in tympanal
structure and mechanics (Hoy and Robert,
1996). Sexual dimorphism has been documented in the ear anatomy of
a small number of praying mantises (Yager,
1999a), bushcrickets (Bailey
and Römer, 1991), flies
(Robert et al., 1994) and
moths (Minet and Surlykke,
2003), and was found to be obvious in cicadas
(Pringle, 1954). Divergences
in frequency tuning between sexes have been recorded in the ascending neurons
of some crickets, bushcrickets, grasshoppers and cicadas
(Gerhardt and Huber, 2002).
However, little information has been made available on potential mechanical
differences between male and female tympana
(Meyer and Elsner, 1997). The
origin of sexual dimorphism at the anatomical, mechanical or neuronal level
may be explained by selective forces and constraints acting differently on the
sexes. Several sex-linked factors can indeed be put forward: (1) sexes are
exposed to different predators (Cardone and
Fullard, 1988; Yager,
1990; Rydell et al.,
1997); (2) prey or host detection is devoted to one sex only
(Lakes-Harlan and Heller,
1992; Robert et al.,
1994); (3) intra-sexual communication has been reduced, or
disappeared in a single sex (Bailey and
Römer, 1991; Mason and
Bailey, 1998); (4) there is production of sex-specific signals in
duetting species (Bailey,
2003); (5) the acoustic role of the sexes in pair formation is
unbalanced; (6) each sex inhabits a specific niche implying different
environmental constraints on sound propagation. To understand how one or
several of these factors work at shaping the structural basis and functional
diversity of insect auditory sexual dimorphism, it is necessary to study a
model that shows an obvious sexual dimorphism and for which acoustic
communication is well known.

In cicada, one of the noisiest animals in the world
(Bennet-Clark and Young, 1992),
there is extreme sexual dimorphism in the sound production system. Males
possess a pair of abdominal tymbals fully dedicated to the generation of the
calling song, a unique system that does not appear in the female
(Pringle, 1954;
Bennet-Clark and Young, 1992;
Young and Bennet-Clark, 1995),
hence the absence of any inter-female acoustic communication. Both male and
female are nonetheless endowed with fully developed tympana whose differences
in size and shape have been recognized since the middle of the nineteenth
century (Dugès, 1838;
Powell, 1873). These tympana
are extended by a cuticular apodeme to which a set of sensory neurons
(scolopidia; type I monodynal receptors) are attached. Tympana can therefore
be considered as the first and necessary step of the mechanical chain that
ensures audition in cicadas. The male tympanum is always larger, and is often
coupled to a large air-filled abdomen. This dimorphism has been associated
with the mechanism of sound radiation through the tympana and abdomen
(Young, 1990;
Bennet-Clark and Young, 1992;
Fonseca and Popov, 1994), but
is undoubtedly involved in different auditory capacities of the sexes. Few
attempts have been made to characterize the effects of dimorphism on auditory
capability. In the Australian bladder cicada, Cystosoma saundersii,
differences in tympanal and abdomen morphology drastically reduce the male's
ability to localize a sound source, whereas the female exhibits accurate
directional sensitivity (Young and Hill,
1977; Fletcher and Hill,
1978). In the Iberian cicada, Tympanistalna gastrica,
larger tympanal membranes have been reported to impart a higher sensitivity to
males (Fonseca, 1993) and in
Cicada barbara lusitanica different tympanal structures imply
different tuning and directionality
(Fonseca and Popov, 1997).
Tympanal membranes of both male and female Cicadatra atra vibrate
with similar travelling waves, but males, with larger tympana, are slightly
detuned to their own calling song, a system that might protect their auditory
sensitivity (Sueur et al.,
2006).

Do the morphological differences in cicada ears imply different auditory
mechanics between sexes? What then could be the origin and consequences and
diversity of sexual dimorphism in cicada audition? We analysed the mechanics
of Cicada orni, an otherwise well investigated species with obvious
sexual dimorphism affecting the hearing system. The histology of the
chordotonal system has been studied previously in detail
(Vogel, 1922;
Michel, 1975) and the
frequency tuning at the auditory nerve level has been measured
(Popov et al., 1992), but
nothing is known about the mechanics of the TM, where sound is transduced into
a mechanical vibration. Using laser Doppler vibrometry, surface deflections of
C. orni TM were reconstructed in three dimensions. This study reveals
different deflection patterns than those previously observed in C.
atra (Sueur et al.,
2006), suggesting that different mechanical processes for
filtering sound frequency content have evolved among cicadas.

MATERIALS AND METHODS

Animals for laser vibrometry experiments

Male and female Cicada orni L. were caught on the 9th July 2007 in
Cuges-les-Pins, France (N43°16′18″ E5°41′24″).
Animals were cooled down to 8–10°C and were immediately transferred
to Bristol, UK in an ice-box. As previously described in detail
(Sueur et al., 2006), animals
were kept at this temperature but placed at 24–26°C before
measurements. The wings, the legs, the operculum and the meracanthus, which
are not mechanically linked to the tympanal organs, were cut back before
mechanical measurements were made. Animals were not anaesthetized during
measurements, but were firmly attached to a horizontal brass bar (6 mm wide, 1
mm thick and 16 mm long) using Blu-Tack (Bostik-Findley, Stafford, UK). The
brass bar was connected to a metal rod (150 mm long, 8 mm diameter)
via a thumbscrew, allowing the animal to be rotated and tilted into
the required position. Only one ear was examined per animal. Tympanal
vibrations were measured with a microscanning laser Doppler vibrometer
(Polytec PSV-300-F; Waldbronn, Germany) with an OFV-056 scanning head. The
animal was orientated such that the measuring Doppler vibrometer could scan
the entire tympanum and that the tympanum was perpendicular to the direction
of sound wave propagation. All experiments were carried out on a vibration
isolation table (TMC 784-443-12R; Technical Manufacturing Corp., Peabody, MA,
USA) at 24–26°C and 40–62% relative humidity. The vibration
isolation table with the animal and the laser vibrometry measurement head were
located in a dedicated acoustic isolation booth (Industrial Acoustics IAC
series 1204A, internal dimensions: length 4.50 m, width 2.25 m, height 1.98
m).

Calling song recordings

The calling song of C. orni could not be recorded in the same
location (Cuges-les-Pins) because of the massive occurrence of two other
singing cicada species, Cicadatra atra and Lyristes
plebejus, that generated an important background noise. A previous study
showed that the calling of C. orni song from western Europe, in
particular from France, constituted an homogenous group
(Pinto-Juma et al., 2005). We
then used previous recordings made in two other locations (Peyriac-de-Mer,
France, N43°05′14″ E2°57′33″;
Molitg-les-Bains, France, N42°39′9″ E2°23′6″),
at other dates (16th and 17th of July 2001) but at the same ambient
temperature (26–27°C) of the sound-acoustic-proof room where laser
experiments were carried out. Recordings were made using a Telinga Pro4PiP
microphone (Telinga Microphones, Tobo, Sweden) (frequency response
40–18000 Hz ±1 dB) connected to a Sony TCD-D8 digital audiotape
recorder (sampling frequency: 44.1 kHz, frequency response flat within the
range 20–20000 Hz). The microphone was held at 50–60 cm dorsally
from isolated singing males. One minute of each male calling song was analysed
in the frequency domain using Seewave
(Sueur et al., 2008). A mean
frequency spectrum with a resolution of 12.5 Hz was computed for each
individual using a Fourier transform with a Hamming window.

Mechanical measurements

The vibrations of the tympanum were studied following the same general
procedure used in a previous study (Sueur
et al., 2006). The vibrations of the whole tympanum were examined
in response to frequency modulated signals (duration=80 ms) sweeping at
similar intensity all frequencies from 1 kHz to 22.05 kHz (low frequencies;
LF), or all frequencies from 20 kHz to 80 kHz (high frequencies; HF). All
acoustic stimuli were amplified with a Sony amplifier model TAFE570 (Tokyo,
Japan) and were broadcast at 0.25 m from the cicada with a ESS AMT-1
loudspeaker (ESS Laboratory Inc., Sacramento, CA, USA) for LF, and with a
SS-TW100ED loudspeaker (Sony) for HF. Thus, for both LF and HF ranges, the
animal was in the far-field of the sound source. The vibrations of the
tympanal ridge (TR), a dark spear-like structure connected to the apodeme
where the sensory neurons (scolopidia) are attached, were studied in greater
detail in six females using a line of scan points. The male TR was not
examined in such a way as it was partially hidden by a cuticle sternal
expansion that could not be removed without damaging the tympanum.

The intensity of the acoustic stimulations was 66 dB SPL at the cicada
position. This corresponded to the sound pressure level (SPL) of a male
calling at a distance of 4 m (Sueur and
Aubin, 2003). This SPL was above auditory nerve threshold
(Popov et al., 1992). The
tympanal and female ridge vibrations were analysed by simultaneously recording
the vibration velocity of the tympanum and the SPL adjacent to the tympanum.
The laser vibrometer allowed accurate measurement (laser positioning ∼1μ
m) of the topography of tympanal motion in the amplitude, time and
frequency domains, in a contact-free way and without requiring the use of a
reflective medium on the TM. SPL was measured using a 1/8 inch (3.2 mm)
precision pressure microphone (Bruel & Kjaer, 4138; Nærum, Denmark)
and preamplifier (Bruel & Kjaer, 2633). The microphone has a linear
response in the measured frequency range. The sensitivity of the microphone
was calibrated using a Bruel & Kjaer sound level calibrator (4231;
calibration at 1 kHz, 94 dB SPL). The microphone was positioned 10 mm from the
tympanum, with its diaphragm parallel to the sound direction, thus maximizing
the response.

The analysis of the tympanum displacement was carried out by the PC
controlling the vibrometer. The laser signals resulting from the FM sweep were
simultaneously sampled at 102.4 kHz for LF and at 204.8 kHz for HF. Sets of 15
data windows of 80 ms duration were acquired and averaged for each point
across the membrane. Using an FFT (Fast Fourier Transform) with a rectangular
window, a frequency spectrum was produced for each signal with a resolution of
12.5 Hz. The laser and microphone signals were then used to calculate
different quantities, such as gain and phase responses. By combining the
results from all the points scanned, oscillation profiles and animations of
tympanal deflections were generated for specific frequencies.

Frequency spectra of the laser signal were normalized to those of the
microphone signal by the computation of transfer functions, calculated as the
cross-power spectrum of the laser and the microphone signals divided by the
auto-power spectrum of the latter. In addition, the amount of unrelated noise
was estimated by calculating the magnitude squared coherence (the ratio
between the squared absolute value of the cross-power spectrum between the two
signals divided by their auto-power spectra). Coherence values can range
between zero and one, with a value of one indicating the absence of external,
unrelated noise. Data were considered of sufficient quality when coherence
exceeded 85%.

Spectral analysis and statistics

To describe both calling song and tympanal frequency spectra, we used a
measure of resonance quality at –3 dB around the dominant peak
(Q–3dB)
(Bennet-Clark, 1999) and an
estimation of spectral flatness (SFM; spectral flatness measure),
which is the ratio of the geometric and arithmetic means of the frequency
spectrum (Jayant and Noll,
1984). Values of Q–3dB increase with
peak sharpness and values of SFM lay between 0 and 1, which
respectively are indicative of a pure-tone signal and a random noise.

Anterior and posterior (after removing the abdomen) views of male and
female C. orni right tympanal membrane (TM). The male TM consists of
three distinct parts, two of them are outlined with blue and green lines,
respectively, and the third part is the area between. Female TM can be divided
in two parts, one of them being shown in red. For both male and female the
ridge area is indicated with a yellow dotted line. The shape of these parts
differs slightly between anterior and posterior views as access and angle of
view to the surface of the TM also differ. Scale bars, 0.5 mm.

RESULTS

Tympanal anatomy

Like many other cicadas, the hearing system of C. orni resides
ventrally in the second segment of the abdomen. The auditory system comprises
two major elements: the tympanum and the sensory organ proper. The tympanum is
a thin membrane of cuticle backed by a tracheal air chamber. The tympanal
membrane (TM) is a heterogeneous structure; it is partially crossed by a dark,
spear-like structure called the tympanal ridge (TR). This ridge is extended by
the tympanal apodeme, hidden in the auditory capsule, where the sensory
neurons are attached. Conspicuous differences in size, shape and thickness are
apparent between male and female tympana. Males have larger tympana than
females surrounded by a larger cuticular frame, the dorsal rim being
significantly stronger (Fig.
1). The male tympanum has three main zones. There are two opaque,
white, thick zones that occupy medially and laterally three quarters of the TM
surface (Fig. 1, blue and green
lines). Between them lie one transparent thin central zone crossed by the
ridge (Fig. 1, yellow dashed
line). The latter is large and short, its apex reaching only one third of TM
width. The female tympanum has only two zones, one is transparent, occupying
three quarters of the TM surface, and crossed by a long, thin ridge, with its
apex reaching around 80% of TM width (Fig.
1, yellow dashed line). The second zone, laterally located, is
darker but not totally opaque (Fig.
1, red line).

Spectral characteristics of the male calling song

The calling song of 10 males were recorded and analysed. The signal
consists of short echemes regularly repeated [for a detailed analysis of the
temporal pattern see Pinto-Juma et al.
(Pinto-Juma et al., 2005)
(Supplementary material Audio 1)]. In the frequency domain, the calling song
covers a wide band, from around 1.5 to 19 kHz with 50% of the energy between
4.46±0.21 kHz (mean ± s.d.) and 6.73±0.74 kHz
(Fig. 2A). The dominant
frequency is 4.5±0.17 kHz with a resonance quality factor,
Q–3dB=10.04±1.66. The peak of the first
frequency band is 2.27±0.18 kHz, its relative amplitude compared to the
dominant frequency being at –17.05±4.19 dB. The signal is not
modulated in frequency but a fast amplitude modulation at a rate of about 1
kHz is present due to the pulsed structure of the signal introducing secondary
peaks every 1 kHz (Fig.
2A).

Frequency magnitude spectra of the male calling song, and of the TM
vibrations of both sexes. (A) Calling song spectra of 10 distinct males (grey
lines) and their mean (black line), 50% of the male calling song energy is
highlighted with a light grey shading. (B) TM vibrations spectra of 11 males
and seven females (thin lines) and their respective mean (bold lines) at low
frequencies (1–22.05 kHz), vertical dotted lines show the correspondence
between maximal TM resonance and male calling song spectra. (C) TM vibrations
spectra of six males and eight females and their respective mean (bold lines)
at high frequencies (20–80 kHz). Originally expressed as amplitude data
(mV for recorded songs) or gain data (nm Pa–1 for TM
vibrations), spectra were normalized between 0 and 1 and then transformed in
decibels (dB) for the purpose of comparison.

Spectral characteristics of male and female entire TM

Scanning the entire TM surface with the laser Doppler vibrometer with
meshes of 151±23 (females), 164±27 (males) points allows the
measurement of the mechanical response of the cicada hearing system in the
frequency domain. Averaging all points measured, it is then possible to obtain
a frequency magnitude spectrum that indicates for which driving frequency the
whole TM vibrates the most and thus reveals the first step of mechanical
filter processes. In the low frequency range (LF, 1–20 kHz), the TM
response of 11 males showed a sharp dominant peak at 2.13±0.30 kHz with
a Q–3dB factor at 2.92±0.86
(Fig. 2B). The concentration of
energy around this dominant peak is confirmed by intermediate SFM
values (0.542±0.072). The male TM is therefore sharply tuned to the
lowest frequency component of the male calling song
(Fig. 2A,B, vertical dashed
blue line). The frequency response between 1 and 20 kHz is broader for the
seven females as shown by significantly higher SFM values at
0.93±0.022 (Welch t-test: t=–16.6096,
d.f.=12.648, P=5.778×10–10)
(Fig. 2B). The dominant peak is
higher at 4.35±0.29 kHz (Welch t-test,
t=–15.655, d.f.=13.456,
P=5.052×10–10) with a similar
Q–3dB at 2.71±1.11 (Welch t-test,
t=0.453, d.f.=10.884, P=0.659). Thus the female TM vibrates
over a wide frequency band, but has a sharp maximal resonance exactly matching
the male's calling song dominant frequency
(Fig. 2A,B, vertical dashed red
line). Displacement gain at the frequency peak is 486±153 nm
Pa–1 for males and 119±46 nm Pa–1 for
females (Mann–Whitney test: W=0,
P=6.285×10–5). At their best resonant
frequency, the male TM is then moving 4.08 (=12.2 dB) times more than female
TM. This partly compensates for the relative amplitude difference between the
2.1 kHz and 4.5 kHz frequency bands of the calling song.

Motion patterns of male and female tympana

Three-dimensional reconstruction of the laser Doppler data reveals the
patterns of motion of the tympanal system
(Fig. 3A). At low frequencies,
notably around the resonance peak at 2 kHz, the entire male TM vibrates in a
simple oscillatory motion (Supplementary material Movie 1). This is
particularly clear when looking at the envelopes of deflection shapes across
the TM (Fig. 3B). The point of
maximum deflection is located at the centre of the TM, close to the apex of
the ridge. When stimulating male TMs at high frequency (50 kHz), only the
transparent middle zone is vibrating, the two other zones remaining still
(Supplementary material Movie 2). In this case the ridge is almost not driven
by the TM, its apex being outside the area of maximal TM motion. The female
tympanal system shows different patterns of motion
(Fig. 3A). Around the frequency
peak at 4 kHz, the female TM moves up and down asymmetrically but in phase.
The lateral opaque zone is notably moving more than the rest of the membrane
(Supplementary material Movie 3). This generates asymmetric deflection shape
envelopes across the TM (Fig.
3B). Lying in the central part of the TM, the ridge is away from
the maximal deflection point. Driven with HF, female TM showed a different
pattern as seen for a 50 kHz stimulus in
Fig. 3 (Supplementary material
Movie 4). The membrane was moving up and down maximally in its middle part
exactly where the ridge is found. This motion is organized, as not all TM
points were moving exactly in phase.

Deflection shapes of a male right TM and of a female left TM. The TM was
stimulated with a FM sweep signal. (A) Oscillations are shown at eight
different phases (45° increment) along the oscillation cycle at the best
resonance frequency in the low frequency domain (2.075 kHz for the male, 4.1
kHz for the female) and at 50 kHz. Deflections are expressed as displacement
gain following the colour scale (nm Pa–1). Red indicates
outward tympanal deflections and green inward tympanal deflections. Note the
difference in scale for each sex, and each driving frequency. Orientation is
indicated by a 3D space reference (P, post; A, ant). The yellow line indicates
the approximate position of the ridge and the green, blue and red lines show
the limits of the different TM parts (see
Fig. 1). (B) Corresponding
envelopes of mechanical deflections (nm Pa–1) across TM along
the W–Z transect line. The position of the ridge apex is indicated by a
vertical yellow line. Green and red curves are minimum and maximum values,
respectively. Note the difference in scale.

Mechanics of the female tympanal ridge

We studied in more detail the mechanics of the tympanal ridge (TR) of six
females. We limited this analysis to the LF domain where frequency
discrimination for male calling song is expected to occur. The male TR is
unfortunately not accessible to the beam of the laser vibrometer. The
differences in TR response with driving frequency are further assessed by
computing the frequency spectrum at each of the measurement points taken along
the ridge. The frequency response of the TR is characterized by two main
peaks, the lowest at 5.53±1.05 kHz (N=155 points for six
females) and the highest at 16.65±2.42 kHz (N=155 points for
six females; Fig. 4A). The
first peak matches 50% of the male calling song spectrum. There is no
frequency modulation along the ridge (Fig.
4B), but the amplitude of the peaks changes from the apex to the
base of the TR (Fig. 4C). When
looking at a normalized frequency response, it appears that the relative
amplitude of the 16.65 kHz peak is maximal and linear along the ridge. Indeed,
this frequency shows the highest relative amplitude for 97.4% of the
measurement points. At the same time, the relative amplitude of the 5.53 kHz
peak is significantly increasing from the apex (0.46±0.15 relative
amplitude, N=6) to the base of the TR (0.83±0.20 relative
amplitude, N=6). However, absolute measurements show that the
displacement of the TR is the same for the 5.53 kHz peak (apex:
102.7±42 nm Pa–1, N=6; base: 97.6±36.2
nm Pa–1, N=6; Welch t-test:
t=0.2249, d.f.=9.786, P=0.8267), but decreases for the 16.65
kHz peak (apex: 223.8±66.4 nm Pa–1, N=6;
base: 127.2±74.3 nm Pa–1, N=6; Welch
t-test: t=2.3752, d.f.=9.876, P=0.03923).
Altogether, this suggests that the TR acts as a low-pass filter: its base is
less sensitive than its apex to frequencies around 16.65 kHz, but is equally
sensitive for frequencies around 6 kHz. Deflection shapes show steady waves
with a drum-like motion, the base of the TR moving less than its apex
(Fig. 5). The phase response
along the TR does not show a significant increasing lag as a function of
stimulus frequency (Fig. 6A).
There is no phase lag either between the apex and the base as shown
(Fig. 6B). This differs
drastically from the phase response of the TR of another species,
Cicadatra atra, in which travelling waves generate phase lags with
both frequency and position along the ridge
(Sueur et al., 2006).

Frequency response of the female tympanal ridge (TR). A line of scan points
was taken along the ridge, and the frequency response was measured at each
point. (A) A typical frequency response of the TR at its apex (red) and at its
base (blue). Both frequency spectra show two main bands, one around 6 kHz, one
around 17 kHz. Grey shading highlights 50% of the male calling song energy to
show the match with the lowest frequency peak. Spectra were normalized between
0 and 1 to allow profile comparison. (B) Frequency of the two main spectra
peaks. Filled circle indicate the lowest frequency peak and open circles, the
highest peak. Different colours indicate different females. The number of scan
points was not the same for all females. Grey area as in A. (C) Variation of
the relative amplitude of both peaks on a linear scale normalized between 0
and 1. The relative amplitude of 17 kHz peak is linear and maximal whereas the
relative amplitude of the 6 kHz peak increases along the ridge.

Deflection shapes along the female TR from its apex to its base. The ridge
was driven at its best resonance frequency in the low frequency domain (4.1
kHz) and at 50 kHz for the high frequency domain. Green and red curves are
minimum and maximum values, respectively.

Phase response of the female TR. (A) Phase response along the ridge from
its apex to its base. (B) Difference between the phase response measured at
the apex and at the base. There is no significant increasing phase lag with
frequency, the maximal difference being 74° at 8.225 kHz. For comparison
similar data measured for a Cicadatra atra female are shown, where
travelling waves occur along the ridge. In this case the phase lag reaches
204° at 19.912 kHz. C. atra data are modified from Sueur et al.
(Sueur et al., 2006).

DISCUSSION

Believing that the cicada hearing system is architecturally homogeneous, we
expected to observe similar tympanal mechanics between C. orni and
C. atra, as both species belong to the same tribe Cicadini. The
vibration pattern of the C. atra membrane is complex, characterized
by waves travelling across the ridge, the phase and wavelength of which vary
with the driving frequency, and with male and female showing slight tuning
differences (Sueur et al.,
2006). Surprisingly, we found significant differences in C.
orni, revealing a further aspect to insect auditory diversity. C.
orni seems to have developed a distinct mechanical strategy that filters
out frequencies not relevant to acoustic communication. The C. orni
tympanum indeed moves with a simple drum-like motion similar to the membrane
of a microphone (Windmill et al.,
2007) and show important sex-specific characteristics, which we
hereafter compare in more detail.

Male acoustic reception

Scanning the whole male TM indicates that the system is designed to receive
one specific frequency band around 2.1 kHz. The resonance of the TM is sharply
tuned around this frequency, and the ridge apex is not driven significantly at
higher frequencies, in particular at ultrasonic frequencies. This tuning seems
to be conserved, but slightly broader, when recording the summed excitation of
the auditory receptors in the auditory nerve
(Popov et al., 1992). However,
more recent intracellular recordings from auditory interneurons of another
species (Tettigetta josei) suggest that the cicada ear uses
differential tuning of the auditory receptors for frequency discrimination
(Fonseca et al., 2000).

Frequency selection is accompanied by high sensitivity as indicated by a
maximal displacement gain around 880 nm Pa–1. In other words,
males seem to be able to listen efficiently to a narrow frequency band centred
around 2.1 kHz. Surprisingly, this selectivity is not congruent with the
maximal song energy around 4.5 kHz, but to the lowest component of the
emission spectrum, some 17 dB lower in intensity. This discrepancy between
emission and reception spectra is probably linked to the large size of the
tympanum, knowing that the frequency of the first mode of vibration is
inversely proportional to the square root of the area of the membrane
(Fletcher, 1992). Such apparent
detuning can, however, confer some advantages. With such a high sensitivity a
perfect tuning with the calling song dominant frequency would probably
overdrive the system during self-generated calling. If the auditory threshold
can be reduced by the tympana folding through the action of an accessory
muscle (Hennig et al., 1994),
frequency detuning may also provide some protection of sensitivity, and
prevent deafening. It is also important to note that a mismatch between
mechanics and calling song might disappear when testing the behavioural
response to stimuli with different frequencies. This is, for instance, the
case of the sibling species C. barbara lusitanica, as the males have
an auditory nerve that responds best to 3–4 kHz tones, but behaviourally
have a more sensitive response to 6 kHz sound
(Fonseca and Revez, 2002). It
would be interesting to conduct playback experiments with C. orni to
know whether a correlation between mechanics and behaviour does exist.

In addition, cicada male tympana work like passive radiators of a simple
Helmholtz resonator, whose cavity is the abdomen and drivers are the tymbals
(Young, 1990). Variation in
tympanal structure is likely to modify the quality of the sound produced.
Sound frequency and energy increase with the size of the tympanum and,
inversely, resonant frequency shifts down when thickness augments
(Bennet-Clark and Young, 1992).
The large size of the male tympana might then facilitate a good transmission
of high frequency sound. By contrast, tympana appear to be particularly thick
in their median and lateral parts and thus probably shift the calling song to
lower frequencies than it would have been with only thinner membranes. Because
they are involved in sound emission and reception in the same time, male
cicada tympana work as dual structures, and as such must be the result of a
trade-off between several sets of selective forces.

Female acoustic reception

The female tympanum is precisely tuned to the dominant frequency of the
calling song, presumably maximizing the detection of the species-specific
song, and its recognition. This sharp tuning is probably the result of sexual
selection forces through female choice. It is highly probable that the
temporal pattern of the song, made of the regular repetition of echemes
(Pinto-Juma et al., 2005),
also participates in song identification as was suggested to occur in C.
barbara lusitanica (Fonseca and
Revez, 2002). As in males, the female TM works like a simple
membrane, but the pattern is asymmetric at low frequencies. For the apex of
the ridge, the deflection is maximal at high frequency. A precise examination
of the TR deflection shape reveals that the response amplitude to high
frequency components decreases in amplitude from the apex to the base. It
appears that the TR works as a low-pass filter focussing low frequency
components, around the dominant frequency of the calling song, to its base,
which is directly connected to the internal apodeme where sensory neurons
attach. Because the TR is a part of the TM and not an independent structure,
the vibrations of the other parts of the TM probably contribute to this
mechanical filter. Again, TR deflections follow a simple oscillatory pattern
very different from the complex travelling waves observed in C. atra,
indicating that these two species use different passive frequency filters.

The resonance quality factor (Q–3dB) around
maximal resonance is similar in male and female tympana, but the spectral
flatness measure (SFM) indicates that the female tympanum has a
broader frequency sensitivity, being able to move significantly at frequencies
higher than 6 kHz. This result indicates that the sensitivity of the female
might then cover the whole spectrum of the male calling song. Females are the
searching sex and need to precisely locate singing males. As shown in C.
barbara lusitanica, which is extremely similar in size and morphology to
C. orni, phase and amplitude differences between left and right
tympana due to diffraction around the body are significant only above 10 kHz
and tympanal directionality also increases with frequency
(Fonseca and Popov, 1997). To
be able to listen to a broad frequency spectrum ensures that the females
receive more cues on the localization of the source. Our data reveal that
female auditory capacity not only encompasses the highest frequency part of
the calling song, but might extend into the ultrasound domain. This aptitude
might also be linked to the small size of the tympanum. Although ultrasound
use has never been reported in cicadas, many insects are known to exploit high
frequency sound for mating (Mason and
Bailey, 1998; Skals and
Surlykke, 1999; Montealegre-Z
et al., 2006; Nakano et al.,
2006) or during prey–predator interactions
(Lakes-Harlan and Heller,
1992; Yack and Fullard,
2000; Ratcliffe and Fullard,
2005; Höbel and Schul,
2007). It is now necessary to conduct behavioural observations and
experiments to determine in which context – reproduction or predator
avoidance – cicadas might use ultrasound. This would also encompass
recordings of auditory neurons to ensure that ultrasound is integrated by the
neuronal system.

LIST OF ABBREVIATIONS

HF

high frequency

LF

low frequency

Q–3dB

resonance quality factor at –3dB

SFM

spectral flatness measure

TM

tympanal membrane

TR

tympanal ridge

ACKNOWLEDGEMENTS

This work was financially supported by a Company of Biologists JEB Travel
Fellowship and by a grant from the British Council in France. We thank Joseph
C. Jackson and three anonymous referees for their helpful comments on the
manuscript. We are indebted to Stéphane Puissant and Sofia Seabra for
their assistance when recording cicada calling song in the field.

Sueur, J. and Aubin, T. (2003). Is microhabitat
segregation between two cicada species (Tibicina haematodes and
Cicada orni) due to calling song propagation constraints?
Naturwissenschaften90,322
-326.

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